The production of biological oils from sources such as plants (including oilseeds), microorganisms, and animals is essential for various purposes. For example, the production of biodiesel requires large quantities of biological oils. Biodiesel has been proposed as a carbon-neutral liquid fuel alternative to petroleum-derived diesel. Biodiesel is most commonly formed by the transesterification of acyl groups of vegetable oil lipids using a simple alcohol (such as methanol, ethanol, or isopropanol). The resulting alkyl esters can then be burned directly in most modern compression ignition engines without any mechanical modifications. Energy density of biodiesel has been estimated at 95% of that for petroleum diesel (or “fossil diesel”). However, the higher lubricity of biodiesel (and thus improved fuel efficiency) results in approximately equal mileage obtained from equivalent volumes of either fossil- or biodiesel.
Since biodiesel is currently made primarily from the seed oils of CO2-fixing plants, the fuel is considered “carbon-neutral” in that all of the CO2 emitted from burning biodiesel was already in the atmosphere recently as opposed to fossil diesel which when burned, releases carbon that has not been in the atmosphere for millions of years. Therefore, biodiesel and other carbon-neutral fuels may have much to contribute to world-wide efforts to reduce the emission of greenhouse gasses (such as CO2).
Several states in the United States have mandated that biodiesel be mixed with fossil diesel sold in that state and the federal government has also set goals for the use of renewable transportation fuel. Current supplies of vegetable oils for conversion to biodiesel have had trouble meeting these mandate levels, resulting in higher prices for many oilseed crops, particularly soybeans. If current trends continue, prices of important oilseed crops could rise significantly. Ultimately the goal is to supplant all sources of fossil fuels with competitively priced bio-based alternatives. Unfortunately, if current sources of oil for biodiesel do not change significantly, this goal may never be realized.
Recognizing this challenge, investigations have been conducted on alternative sources of oil for biodiesel production, including the feasibility of making biodiesel from photosynthetic algae grown in open ponds. Since some algae are oleaginous and grow very quickly (for some, the duration of time from inoculum to harvest is less than two weeks), the theoretical yield of oil per acre per year could be orders of magnitude greater than what could be derived from higher plants. It should be noted that the small portions of the seeds of most oil-producing higher plants represent only a small fraction of the overall mass of the plant, whereas photosynthetic microalgae might accumulate a higher percentage of their mass as oil useful for biodiesel production. There are however, serious problems with the photosynthetic algae technology that prevent the massive scale-up which are required to compete effectively with fossil diesel technology.
The photosynthetic microalgae often had to be supplemented with CO2 in order to achieve high yields of oil. From the perspective of bioremediation, this is actually a benefit as excess CO2 released from coal or oil-fired electrical plants, which would otherwise be released to the atmosphere, could be used as a feedstock for making biodiesel. This approach obviously does not produce a truly carbon neutral fuel as the CO2 from a coal plant is still released to the atmosphere eventually (after the biodiesel is burned), but it does delay the rate at which fossil-derived CO2 is released and generates more useful energy per unit mass of fossil fuel. In fact, several companies have been established to capitalize on this technology, including Greenfuels Inc. Greenfuels specifically uses closed photobioreactor systems which dissolve very high levels of CO2 from fossil-fuel-fired electrical plants into photosynthetic algal cultures. Due to the biophysical limitations of self-shading, accumulation of biomass is dependent upon total illuminated surface area. Thus, many photobioreactors are required to produce even limited quantities of biodiesel. Therefore, while this technology is useful as a bioremediation strategy for sequestering carbon (and other greenhouse gases) from fossil-fuel burning electrical plants, it is unlikely scalable to the levels required to meet future biodiesel demands.
To address the issues of scalability, other organizations have opted to further develop open pond technologies for making phototrophic, algal-derived biodiesel. Open pond systems also rely on CO2 supplementation for hypothetically economical levels of oil accumulation. Therefore these systems also may be better regarded as systems for bioremediation of waste carbon from fossil fuels. The yields per acre per year of useful oil from these systems are orders of magnitude greater than what can be derived from seed-oil crops. From most perspectives, these systems appear to be the best answer to limited supplies of biodiesel oil. However, there is a significant problem which has not yet been addressed. While the absolute theoretical yields of oil per acre per year are quite high, the actual density of biomass accumulated in open pond systems is relatively dilute. Because of this, massive volumes of culture media need to be processed to extract the oil from the biomass, which could significantly increase the costs of the final oil.
A path to the replacement of gasoline with renewable alternatives such as ethanol is less complex. It should be noted, however, that the markets for compression combustion engines (which burn fossil diesel or biodiesel) and for ignition combustion engines (which burn gasoline or ethanol) generally serve different needs. Compression ignition engines offer superior torque, which make them more useful in industrial applications over ignition combustion engines, which offer greater acceleration (thus making the latter more popular for general commuting). Hence, there is no reason to expect that the ignition combustion engine could fully replace the compression combustion engine should a renewable replacement for gasoline ever be fully adopted.
Despite certain disadvantages, much has been made of the potential for ethanol to supplant gasoline as a liquid transportation fuel. The Brazilian model, which relies on sugarcane as a feedstock for ethanol fermentation, has been often cited as a pioneering example for bio-fuel viability. Unfortunately, the United States does not have a climate that could support the kind of sugarcane productivity needed for massive ethanol production. Initial efforts at scaling up American ethanol fermentation have used corn syrup and corn starch as a feedstock, but there is controversy surrounding the sustainability and scalability of this arrangement as well. Because of this, more recent efforts have focused on “cellulosic” sources of sugars to use as feedstocks in ethanol fermentation. Cellulosic feedstock can be any feedstock containing cellulose.
Because most plants are primarily composed of structural polysaccharides (cellulose and hemicellulose) and lignan, acreage can be used more efficiently if the sugar monomers of cellulose and other structural polysaccharides are mobilized as a feedstock for ethanol fermentation. This is in contrast to using corn starch, which is found only in the corn plant kernels and constitute a relatively low percentage of the crop's dry weight. Additionally, since all plants contain cellulose, much faster-growing and more climate-tolerant plants can be used as the primary source of cellulose-based sugar. Examples of such plants include Switchgrass, Miscanthus gigantus, and Poplar.
Today's primary biodiesel crops use land in a similarly inefficient way (as corn for ethanol) since only the oil from the seeds of biodiesel crops is used to make biodiesel. Cellulosic ethanol processes have yet to be adopted on a broad scale but thus far cellulosic ethanol is widely accepted as a possible sustainable and economically competitive alternative to gasoline. Cellulosic feedstocks are already being considered for the manufacture of other petroleum-derived products (like plastics).
Patent application publication nos. WO 2005/035693, US 2005/0112735, WO 2007/027633, WO 2006/127512, US 2007/0099278, US 2007/0089356, and WO 2008/067605, the contents of which are incorporated herein by reference in their entirety, all relate to biodiesel or biofuel production systems.
Recently, heterotrophic growth of the microalga Chlorella protothecoides by fermentation has been investigated for purposes of biodiesel production. Researchers at Tsinghua University in Beijing, China have performed studies on biodiesel production using oil from the heterotrophic microalga Chlorella protothecoides. In these studies, microalgae are grown in fermentors using glucose or corn powder hydrolysate as sources of carbon. Microalgal oil is then extracted and transesterified to produce biodiesel. See Miao, X. and Wu, Q., Bioresource Technology 97: 841-846 (2006); Xu, H. et al., Journal of Biotechnology 126: 499-507 (2006). Although these researchers have suggested that starch and cellulose hydrolyzed solutions can be a low cost substitute for glucose as a carbon source in the fermentation process, they have also suggested that cellulose hydrolyzation is difficult and costly. See Li, X. et al., “Large-scale biodiesel production from microalga Chlorella protothecoids through heterotrophic cultivation in bioreactors,” Biotechnology and Bioengineering, Accepted Preprint, Accepted Apr. 20, 2007.
In addition to diesel, another oil-based fuel that is in need of a renewable and sustainable source is jet fuel. Aircrafts depend on the use of various types of jet fuels, including kerosene-type jet fuels and naphtha-type jet fuels. The heavy reliance of the aviation industry on the limited supply of petroleum-based jet fuels creates an urgent need for the discovery of renewable jet biofuels.
Therefore, there exists a need for a low-cost and efficient method for producing lipid-based biofuels that can be easily scaled up to replace fossil diesel and jet fuels. As used herein, “lipid-based biofuel” refers to any fuel that is produced from a biological oil of the present invention, including, but not limited to, biodiesel, jet biofuels, and specialty fuels. In order to satisfy this need, an inexpensive and simple method must be developed for producing biological oils which can be converted to lipid-based biofuels. To reduce the costs of lipid-based biofuels production, there exists a need for a low-cost method of producing biological oils through the use of abundant and inexpensive raw materials, such as cellulose-containing feedstock as a main carbon source. In addition to a need to use inexpensive raw materials, there exists a need for improved processes that also target cost reduction in the production of biological oils. The improved methods of producing these biological oils will not only lower the cost of lipid-based biofuels production, but will also reduce the costs associated with the use of these biological oils in many other applications, including food, nutritional, and pharmaceutical products.
For example, it is desirable to increase the dietary intake of many beneficial nutrients found in biological oils. Particularly beneficial nutrients include fatty acids such as omega-3 and omega-6 long chain polyunsaturated fatty acids (LC-PUFAs) and esters thereof. Omega-3 PUFAs are recognized as important dietary compounds for preventing arteriosclerosis and coronary heart disease, for alleviating inflammatory conditions and for retarding the growth of tumor cells. Omega-6 PUFAs serve not only as structural lipids in the human body, but also as precursors for a number of factors in inflammation, such as prostaglandins, leukotrienes, and oxylipins. Long chain omega-3 and the omega-6 PUFAs represent important classes of PUFAs.
There are two main series or families of LC-PUFAs, depending on the position of the double bond closest to the methyl end of the fatty acid: the omega-3 series contains a double bond at the third carbon, while the omega-6 series has no double bond until the sixth carbon. Thus, docosahexaenoic acid (“DHA”) has a chain length of 22 carbons with 6 double bonds beginning with the third carbon from the methyl end and is designated “22:6 n-3”. Other important omega-3 LC-PUFAs include eicosapentaenoic acid (“EPA”), which is designated “20:5 n-3,” and omega-3 docosapentaenoic acid (“DPA n-3”), which is designated “22:5 n-3.” Important omega-6 LC-PUFAs include arachidonic acid (“ARA”), which is designated “20:4 n-6,” and omega-6 docosapentaenoic acid (“DPA n-6”), which is designated “22:5 n-6.”
Because humans and many other animals cannot directly synthesize omega-3 and omega-6 essential fatty acids, they must be obtained in the diet. Traditional dietary sources of PUFAs include vegetable oils, marine animal oils, fish oils and oilseeds. In addition, oils produced by certain microorganisms have been found to be rich in LC-PUFAs. In order to reduce the costs associated with the production of dietary sources of PUFAs, there exists a need for a low-cost and efficient method of producing biological oils containing PUFAs. To lower the costs of PUFA containing biological oils, there exists a need to develop a method of producing these biological oils using inexpensive raw materials (such as cellulose-containing feedstock) and improved processes that are designed to lower the costs of production.